mark lindsay ucur
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A HIGH-THROUGHPUT LINEAR EXPRESSION TEMPLATE SYSTEM
FOR ANALYZING PROTEIN ACTIVITY AND STABILITY
MARK LINDSAY, JEFFREY WU, BRADLEY C. BUNDYDept. of Chemical Engineering
Brigham Young University
Utah Conference of Undergraduate ResearchFebruary 28, 2014
What are proteins?
Basic building blocks of lifeNecessary for body functions
Metabolism Immune system DNA
Made up of amino acids
Bioengineering Proteins
Protein research has already improved Catalysts Drugs Vaccines
Protein production market valued at over $100 billion annually.
Unnatural Amino Acids (UAA’s)
20 universal amino acidsMore combinations = more biomedical,
pharmaceutical, and catalysis products
Problems with UAA incorporation
1- Difficulty of Incorporation2- High time cost3- Effects on protein structure
Simplifying UAA Incorporation: Cell free protein Synthesis
N3
Cu(I) N NN
DNA of Protein of Interest, Amber
Codon Replacement
UAA tRNA Synthetase
UAA
LyseLysis
Add
Linear Expression TemplatesImproving Time Efficiency
Polymerase Chain Reaction (PCR)`
(http://users.ugent.be/~avierstr/principles/pcr.html)
Protein Structure
Protein activity is highly dependent on structure
Changing Amino Acids can significantly affect structure
Example: Sickle Cell Anemia
Immobilized Enzyme Activity Data
0 10 20 30 40 50 600
5000
10000
15000
20000
25000Random Attachment (at primary amines)
Attachment Near Active Site (T21Amb)
Attachment Near Active Site (K35Amb)
Time (min)
Flu
ore
sc
en
ce
(rf
us
)
Current Work: High-Throughput Testing
Create UAA mutations at dozens of selected sites
Simultaneously test effect of UAA incorporation on: Protein activity Protein stability
Acknowledgements
Research Advisor Dr. Brad Bundy
Graduate Students Jeff Wu
Undergraduates Chris Werner Chris Hutchings Steven Stanley Justin Crandall
Funding DARPA YFA NSF CAREER NASA NPB BYU
Bundy Lab Presentations at UCUR
Bennett A, Smith MT, Bundy BC. Foot-and-Mouth Disease Vaccine: Technical and Political Challenges to Vaccine-based Eradication. Utah Conference on Undergraduate Education. Brigham Young University. Feb 2014.
Hunt J, Smith MT, Bundy BC. Cell-free Unnatural Amino Acid-Incorporation using Linear Expression Templates. Utah Conference on Undergraduate Education. Brigham Young University. Feb 2014.
Hutchings C, Wu JC, Bundy BC. Immobilizing Biocatalysts onto Surfaces. Utah Conference on Undergraduate Education. Brigham Young University. Feb 2014.
Werner C, Bundy BC. Investigating Linear DNA Expression Templates. Utah Conference on Undergraduate Education. Brigham Young University. Feb 2014.
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